SUMMARY (PROJECT 1) The four dengue virus serotypes (DENV1-4) cause the most important mosquito-borne viral disease of humans, with ~100 million cases annually and over 3 billion people worldwide at risk of infection. A major challenge in development of vaccines is that whereas infection with one DENV serotype leads to durable immunity against the same serotype, subsequent infection with a different (heterotypic) serotype is the major risk factor for severe disease. The mechanisms by which the host immune response to DENV provides either protection or enhancement in secondary (2) infection are poorly understood. However, the vast majority of 2 DENV infections are asymptomatic, suggesting an effective protective response can be mounted. The overall approach of Project 1 is to take advantage of unique sample sets from long-term ongoing studies of dengue in Nicaragua to address complex questions about dengue B cell immunology in a relevant epidemiological context. It is designed so that the data generated from characterization of the antibody (Ab)/B cell immune profile can be (1) analyzed in relation to infection outcome and disease severity in Project 1; (2) combined with CD4+ and CD8+ T cell responses in the same sample sets (Project 3) to identify immune correlates of protection in natural infections (Core C), and (3) compared to vaccine-induced Ab/B cell responses and correlates of protection obtained using the same methodologies (Project 2). Each aim also addresses key questions in dengue immunology using both polyclonal Ab analysis and monoclonal Abs. The overall hypothesis is that the protective Ab/B cell response is fundamentally different after a 1 vs. a 2 DENV infection, and that it is the quality as well as the quantity of neutralizing Abs that determines the protective efficacy. Moreover, we posit that strongly serotype cross-neutralizing Abs after a 2nd infection are derived from existing memory B cell (MBC) precursors and target novel epitopes (Core B). Specifically, we hypothesize that neutralizing Abs that are type-specific and directed to the hinge region of the envelope protein predominate after a 1st DENV infection, whereas the neutralizing Ab response is directed to serotype cross-reactive epitopes after a 2nd and 3rd infection (Aim 1). Next, we hypothesize that the quality (e.g., epitope repertoire), breadth, and magnitude of the pre-existing neutralizing Ab/B cell response will influence the outcome of subsequent DENV infection (disease versus no disease) (Aim 2). Finally, during an acute 2 infection, we hypothesize that the quality (epitope specificity and cross- reactivity) and magnitude of the B cell and neutralizing Ab response will correlate with protection against severe disease (Aim 3). To test these hypotheses, we will characterize the serum Abs and MBC profile after a 1st, 2nd or 3rd DENV infection, prior to a documented subsequent DENV infection, and during an acute 2 heterotypic DENV infection. These studies should provide insight as to the B cell/Ab response after natural DENV infections and generate much-needed immune correlates of protection.